Squalene (2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene), a dehydro-triterpenic hydrocarbon (C30H50) with six double bonds as shown in Chemical Formula 1, is one of the terpenoid compounds forming various chemicals and a precursor of all steroids in plants and animals.
It has been revealed that squalene can effectively inhibit chemically induced skin, lung, and colon tumorigenesis in rodents (Aioi A et al., 1995, Int. J. Pharm., 113, 159-164), and supplementation of squalene-containing diets can activate nonspecific immune functions, affect the absorption efficiency of cholesterol, and lower LDL (low-density lipoprotein) and triglyceride levels. Squalene, the main component of skin surface polyunsaturated lipids, shows antioxidant and moisturizing effects on skin, and thus can be used as a raw material for cosmetics. Recently, it has been also approved for use in drug delivery, in particular, as a vaccine adjuvant (Tritto E. et al., 2009, Vaccine, 27, 3331-3334).
Up to the present, the major commercial source of squalene has been liver oils of deep-sea sharks and certain plant seed oils. However, the continuous supply of the liver oils of deep-sea sharks is in doubt because of environmental pollutants such as heavy metals, concerns over the preservation of marine wildlife, and the supply of the plant seed oils (e.g., olive oil 7 mg/g, amaranth oil 0-5.64 mg/g) is also in doubt because of the effects of seasonal change and geographical variations on the crop production.
Therefore, many studies have been made on the methods for producing squalene, including on the use of squalene-producing yeasts such as Pseudozyma (Chang M H et al., 2008, Appl. Microbiol. Biotechnol., 78, 963-972), Candida famata (JP 07,289,272), and Torulaspora delbrueckii (Bhattacharjee P. et al., 2001, World J. Microbiol. Biotechnol., 17, 811-816), the use of Euglena (JP 07,115,981), and the use of microalgae such as Botryococcus braunii (Okada S. et al., 2000, Arch. Biochem. Biophys., 373, 307-317) and thraustochytrids (Li Q et al., 2009, J. Agric. Food Chem., 57, 4267-4272).
However, these methods do not show sufficient productivity required for commercialization, because of the long period of time required for cell cultivation, low cell productivity, and low squalene yield per cell. Thus, studies have focused on metabolic engineering technologies for high production of squalene.
Saccharomyces cerevisiae has been frequently used as a host cell in metabolic engineering. This yeast has been known to accumulate ergosterol to 1% per dry cell weight on average, and up to 4.6% per dry cell weight depending on the strain or culture conditions (Arnezeder C. et al., 1990, Biotechnol. Lett., 12, 277-282), and thus can be used for the production of squalene that is an intermediate of the ergosterol biosynthetic pathway. However, the accumulation of squalene is very low under normal growth conditions, because it is an intermediate of the ergosterol biosynthetic pathway. That is, squalene productivity of Saccharomyces cerevisiae and Torulaspora delbrueckii is at most 41.16 and 237.25 μg per gram of wet cell, respectively (Bhattacharjee P. et al., 2001, World J. Microbiol. Biotechnol., 17, 811-816), and another recent study revealed that Saccharomyces cerevisiae (BY4741 and EGY48 stains) showed low productivity of approximately 3 mg/l and 3.1 mg/l under semianaerobic conditions (Mantzouridou F. et al., J. Agric. Food Chem. 57, 6189-6198 (2009).
Therefore, metabolic engineering technologies are needed for high accumulation of squalene in yeast. Ergosterol and squalene are isoprenoid or terpenoid compounds. Isoprenoid biosynthetic pathways are largely classified into mevalonate and non-mevalonate pathways, and the mevalonate pathway is ubiquitous in eukaryotes. Because the yeast Saccharomyces cerevisiae is a eukaryotic cell, the mevalonate pathway is ubiquitous therein. The ergosterol biosynthetic pathway is as shown in FIG. 2.
In the mevalonate biosynthetic pathway, the HMG (hydroxymethylglutaryl CoA reductase) step of producing mevalonate is known to be the most important rate-determining step. The step is under various regulatory mechanisms, and overexpression of the HMG gene leads to enhancement of the isoprenoid biosynthetic metabolism. Thus, there have been many attempts to overexpress the HMG gene for excessive production of various isoprenoids (farnesol), geranylgeraniol, and amorphadiene in Saccharomyces cerevisiae (WO2008039499; U.S. Patent No. 20040235088; U.S. Patent No. 20030092144; Tokuhiro K. et al., 2009, Appl. Environ. Microbiol., 75, 5536-5543; Ohto C. et al., 2009, Appl. Micorbiol. Biotechnol., 82, 837-845). It has been known that this method is also useful for squalene production, and overexpression of the HMG1 gene accumulates squalene up to 0.9% per dry yeast cell weight (Polakowski T. et al., 1998, Appl. Microbiol. Biotechnol., 49, 66-71), or 2% per dry yeast cell weight (Donald K A et al., 1997, Appl. Environ. Microbiol., 63, 3341-3344). In addition, a recent study has reported that squalene productivity of 106-192 mg/l (2.3-4.1 mg/g DCW) can be achieved by overexpressing the HMG1 gene in Saccharomyces cerevisiae YPH499 using the GAPDH promoter (Tokuhiro K. et al., Appl. Environ. Microbiol. 75, 5536-5543 (2009)).
Meanwhile, another method for squalene accumulation is to disrupt a gene required for the conversion of squalene to ergosterol by homologous recombination, resulting in accumulation of approximately 5 mg/g of squalene (Kamimura N. et al., 1994, Appl. Microbiol. Biotechnol., 42, 353-357). Ohto et al. recently examined the overexpression effects of various genes involved in the mevalonate pathway (ERG10, HMG synthase, HMG1, ERG12, ERG8, ERG19, IDI1, idi, ERG20, ispA) on the productivities of prenyl alcohols such as farnesol, nerolidol, and geranylgeraniol, and squalene in Saccharomyces cerevisiae (Ohto C. et al., 2009, Appl. Micorbiol. Biotechnol., 82, 837-845), showing that HMG1 was the most effective. However, the ispA (E. coli-derived farnesyl pyrophosphate synthase) gene, which is known to increase the productivity of prenyl alcohols up to 5-6 fold in E. coli, did not show any effects on the improvement of productivity when overexpressed in Saccharomyces cerevisiae (Ohto C. et al., Biosci. Biotechnol. Biochem. 73, 186-188 (2009)). The Erg20 gene, which is the yeast-derived farnesyl pyrophosphate synthase gene, also showed poor effects when overexpressed. The present inventors supposed that the isoprenoid biosynthetic pathway of the eukaryotic yeast is the mevalonate pathway, unlike the non-mevalonate pathway in the prokaryotic E. coli, and an accumulation of farnesyl pyrophosphate as the product of ispA or Erg20 enzyme in the mevalonate pathway inhibits HMG1 gene expression or its enzymatic activity as a regulatory step in the overall isoprenoid biosynthetic pathway.
Accordingly, the present inventors have made many efforts to develop a modified yeast strain for the production of squalene with high efficiency. As a result, they found that the GAL10 or ADH1 promoter can be used as a promoter for the HMG1 gene expression instead of its own promoter to overcome the inhibition of the HMG1 gene expression by the products of isoprenoid biosynthetic pathways and to develop a method of co-overexpressing the farnesyl pyrophosphate synthase gene such as ispA and Erg20, and the HMG1 gene, and the developed modified yeast strain, Saccharomyces cerevisiae Y2805, shows higher productivity by several- to several-tens-fold, compared to the YPH499 strain known to produce squalene with high efficiency, thereby completing the present invention.